The objective of this proposal is to elucidate the role of post-synthetic methylation in gene expression and DNA replication in eukaryotic cells based on our observations 1) that induction of globin synthesis in Friend erythroleukemia cells (FLC) is associated with hypomethylation of the DNA; and 2) that ethionine, an amino acid analogue whose major metabolite inhibits a number of methyl-transfer reactions, not only acts to induce globin synthesis in FLC but reversibly blocks mitogen-induced DNA synthesis in human lymphocytes. The primary aims of the proposed studies are: 1) to characterize the sites of DNA methylation associated with expression of the globin genes and to determine whether such sites are physically linked to the structural genes for globin; 2) to determine whether methylation of other macromolecules is altered during differentiation; 3) to determine which of the macromolecules involved in DNA replication requires methylation for proper function by examining the fate of histones, RNAs and DNA in the presence of and after the removal of ethionine; and 4) to establish, by extending these studies to other cell types, whether a causal relationship exist between gene expression and DNA methylation. The significance of achieving these goals would be, not only an increase in the understanding of the mechanisms of gene regulation, but the first demonstration of a role for the numerous methylated cytosine residues in mammalian DNA.